In Vitro Transcription

3.1.1. DNA Preparation

The detection of gene expression by in situ hybridization is carried out with RNA probes. Efficient RNA transcription requires the subcloning of the DNA fragment of interest into a vector. The multiple cloning site is flanked by phage-RNA-polymerase promoters—for example, T3-, T7- or SP6-RNA polymerase, such as pBluescript II (Stratagene) or, for polymerase chain reaction products, pGEMTeasy, respectively (Promega; Fig. 1A,B). The size of the cloned fragment should range between 0.5 and 3.0 kb. RNAs transcribed from templates larger than 3 kb may be too big to penetrate the tissue, resulting in no signal at all. Conversely, fragments that are too small may cause unspecific background staining. The following procedure explains how to prepare the DNA template used in transcription reactions. All solutions have been either treated with DEPC or prepared with DEPC-treated H2O.

1. Restrict the DNA for 2 to 3 h with an appropriate enzyme cutting the multiple cloning site opposite to the transcription start site (e.g., T7 or T3) used later for in vitro RNA transcription (see Note 16).

2. Extract the restricted DNA with phenol/chloroform. Spin for 5 min in a microcentrifuge and transfer the upper phase to a new Eppendorf tube. Avoid touching the interphase.

3. Extract with chloroform to remove any residual phenol. Spin for 5 min and transfer the top layer to a new Eppendorf tube.

4. Precipitate the DNA with 0.1 vol of 3 M sodium acetate, pH 5.2, and 2.5 vol of 100% ethanol (-20°C) for 30 min at -80°C.

5. Spin for 20 min at maximum speed at 4°C.

6. Remove the supernatant and wash the pellet with -20°C 70% ethanol.

7. Spin for 5 min at 4°C, remove the ethanol, and air-dry the pellet.

8. Resuspend the pellet in DEPC-treated H2O to a final concentration of 1 ^g/^L. Store the DNA at -20°C; it is stable for at least 1 yr.

3.1.2. RNA Transcription

We perform the transcription reaction, following the instructions of the manufacturer of the DIG labeling mix (Roche), except that we use an amount of template equivalent to 1 ^g of insert rather than to 1 ^g of total DNA (vector

I HifK I

GCGCGCG TAATACG« T CAC TATA<KGCGMT TGGG TACCfiGGXC OCC CTGGAGGTCGAC

I HifK I

GCGCGCG TAATACG« T CAC TATA<KGCGMT TGGG TACCfiGGXC OCC CTGGAGGTCGAC

BoalUb I 1'

Oil HmJIli MV t„(l Pal Smt.1 BomH t I ||°91 tJIlS-«» J«l

GO 7-ATC GATAAGC T TGATATC6AAT TCCT GCAGCC GGGGGGATCC ACTAG TTCTAGAGCGGCCGOCACCGC GG T GGAGCTC

Tipromolir- BuHIt

CAGCT TTTGT TCC C TTTAGTGAGGGTTMT TGCGC

Tipromolir- BuHIt

CAGCT TTTGT TCC C TTTAGTGAGGGTTMT TGCGC

Fig. 1. Cloning vectors suitable for in vitro transcription. (A) pBluescript II KS (Stratagene). (B) pGEMT-easy (Promega).

+ insert). This improves the yield of the transcription, and up to 10 ^g of RNA can be generated in a standard reaction.

1. Set up the 20-^L transcription reaction in a sterile Eppendorf tube on ice by adding the following components:

a. x ^L of linear DNA, equivalent to 1 ^g of insert.

b. 4 ^L of 5X transcription buffer.

e. 2 ^L of 10X DIG or Biotin UTP RNA labeling mix.

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